The ability to repair double-stranded DNA breaks (DSBs) by recombination is a universal feature of both prokaryotes and eukaryotes. In addition, mitotic recombination is an important mechanism in the generation of a tumor cell, since recombination can lead to loss of the wild-type allele of a tumor suppressor gene in a cell that is heterozygous for such a mutation. The goal of the proposed studies is to understand the mechanisms of mitotic recombination. Because of the universality of recombination and DSB repair, our experiments, which will be done with the yeast Saccharomyces cerevisiae, will improve our understanding of recombination in higher eukaryotes, including humans. The first Specific Aim is to characterize spontaneous mitotic recombination events. Because mitotic events are 104-fold less frequent than meiotic exchanges, the mechanism of mitotic recombination has been investigated much less extensively than meiotic recombination. We recently developed a novel genetic method of selecting reciprocal mitotic crossovers. We will use this method, coupled with microarray technology, to make the first high-resolution genetic map based on mitotic recombination (Specific Aim IA and ID). This analysis should identify regions of high (hotspots) and low (coldspots) exchange. We will determine whether mitotic hotspots and coldspots correlate with meiotic hotspots and coldspots (Specific Aim IB). We will also use this system to test whether certain DNA sequences stimulate crossovers (Specific Aim IC). One of the sequences that we will test (poly AAG) is associated with the human triplet-repeat-expansion disease Friedreich's ataxia. Our second Specific Aim is to investigate the regulation of spontaneous mitotic recombination events. We will examine the proteins required to catalyze reciprocal crossovers, as well as the proteins required to regulate their frequency. The third Specific Aim is to map DNA damage-induced mitotic crossovers at high resolution, both events induced by ultraviolet light and by gamma radiation. The analysis of DNA-damage induced mitotic crossovers will be done non-selectively throughout the genome, the first such analysis done in any organism.